Locomotor behavior and hearing sensitivity in an early lagomorph reconstructed from the bony labyrinth

Abstract The structure of the bony labyrinth is highly informative with respect to locomotor agility (semicircular canals [SCC]) and hearing sensitivity (cochlear and oval windows). Here, we reconstructed the agility and hearing sensitivity of the stem lagomorph Megalagus turgidus from the early Oligocene of the Brule Formation of Nebraska (USA). Megalagus has proportionally smaller SCCs with respect to its body mass compared with most extant leporids but within the modern range of variability, suggesting that it was less agile than most of its modern relatives. A level of agility for Megalagus within the range of modern rabbits is consistent with the evidence from postcranial elements. The hearing sensitivity for Megalagus is in the range of extant lagomorphs for both low‐ and high‐frequency sounds. Our data show that by the early Oligocene stem lagomorphs had already attained fundamentally rabbit‐like hearing sensitivity and locomotor behavior, even though Megalagus was not a particularly agile lagomorph. This is likely because Megalagus was more of a woodland dweller than an open‐habitat runner. The study of sensory evolution in Lagomorpha is practically unknown, and these results provide first advances in understanding the primitive stages for the order and how the earliest members of this clade perceived their environment.


| INTRODUC TI ON
The middle and inner ear structures in mammals are almost completely enclosed by bone and thus often very well preserved in fossils, even if the rest of the skull is poorly or not at all preserved (Meng & Fox, 1995). Specifically, the bony labyrinth (housing the inner ear) in mammals has been employed in both broad comparative and functional anatomical studies (e.g., Berlin et al., 2013;Ekdale, 2013;Gunz et al., 2012) as well as in more specialized research (see below). The ecological importance of the labyrinthine morphology stems from its potential to be informative about the animal's hearing sensitivity, sense of balance, and locomotor agility, all of which directly influence an animal's lifestyle and behavior.
Cranial material of fossil lagomorphs that predates the Oligocene is extremely rare. The only species known from a partial skull is Dawsonolagus antiquus from the lower part of the Arshanto Formation (late early Eocene) of Nei Mongol, China; however, the skull lacks the posteroventral part, including the ear region (Li et al., 2007). Following the first radiation of the group in the early middle Eocene of Central Asia (Fostowicz-Frelik et al., 2015), lagomorphs quickly appeared in North America, where they have been present since the middle Eocene (ca. 42 Ma, late Uintan North American Land Mammal Age [NALMA], see Dawson, 2008). By the latest Eocene (Chadronian NALMA), North American lagomorphs became quite abundant (e.g., Dawson, 2008), diverging into few distinct lineages, Megalagus, and especially Palaeolagus, being the most common and widespread (Fostowicz-Frelik, 2013).
Concerning the comprehensive anatomy of the bony labyrinth in extant lagomorphs, only the inner ear structures of Oryctolagus cuniculus have been studied in detail (Abd El-Hameed et al. (2023) for CT and MRI imaging; Wysocki et al. (2007) for the topographical anatomy of the temporal). Recently, the first bony labyrinth for a fossil lagomorph (Palaeolagus haydeni, an early Oligocene species) has been described (Ruf et al., 2021). However, Megalagus is a member of a more basal lineage of early lagomorphs (Fostowicz-Frelik & Meng, 2013; see also López-Torres et al., 2020) and the earliest lagomorph for which the structure in question is known, making it of arguably greater relevance to understanding primitive stages for the order.
In this paper, we use high-resolution X-ray CT data to provide the description of a digital endocast of the inner ear of the early lagomorph Megalagus turgidus and reconstruct the locomotor agility and hearing sensitivity of this extinct species compared with those of modern lagomorphs.

| MATERIAL S AND ME THODS
Our study focuses on the otic region of Megalagus turgidus, reconstructed using CT data of an almost complete cranium (FMNH UC 1642) from the early Oligocene (early Orellan), Brule Formation of Grime's Ranch, Sioux County, Nebraska (Dawson, 1958;Olson, 1942 TIFF images of the CT data were visualized in ImageJ (Schneider et al., 2012) and cropped around the bony labyrinth for each specimen using WACOM Cintiq 21UX tablet. The data were resliced using Avizo® 7.0.1 (Visualization Sciences Group, 1995 software so that each semicircular canal (SCC) could be visualized in a single plane (Figure 1; see also Spoor et al., 2007). Images of the cross sections were further analyzed and measured (height and width for each SCC) in ImageJ. We used the better preserved right inner ear endocast for the full reconstruction ( Figure 1). The bony labyrinth structure of Megalagus was further compared with data from extant lagomorphs (leporids and ochotonids), and a variety of modern and extinct Glires (see Figures 1-3; Table 1; for raw data see Appendix A).
We estimated the locomotor agility of Megalagus using an agility score, which was calculated following equations provided by Spoor et al. (2007) and Silcox et al. (2009). The latter paper presented regression equations to calculate agility scores for mammals based on each SCC radii (ASR, PSR, and LSR) as well as an equation based on the average radius for the three SCCs.
According to Silcox et al. (2009), the radius of the lateral semicircular canal (LSC) is the best predictor of agility level, probably because the LSC is the least constrained by the size and morphology of the petrosal lobule (Jeffery et al., 2008). Therefore, we are calculating agility scores based on the radius of the LSC.
The agility score of Spoor et al. (2007) ranges in scale from 1 to 6, with one being extremely slow and six fast animals. Although agility scores for the lagomorph specimens in our sample (see Appendix A: Table A1) are calculated considering the qualitative approach used by Spoor et al. (2007) in assigning agility categories, we also examine data directly through bivariate plots of log 10 LSR versus log 10 BM (BM, body mass) for the combined sample of our new lagomorph specimens and Spoor et al. (2007) lagomorph data (Appendix A).
Previous research on the functional morphology of the auditory system in living euarchontans (Coleman, 2007; found a strong linear relationship between cochlear length (CL) and sound pressure level (SPL) at 250 Hz, and a strong, but less so association between the oval window area (OWA) and SPL at 32 kHz. CL and OWA were estimated by measuring the outer circumference of the cochlear canal and the major (M) and minor (m) axes of the oval window, respectively, following Coleman and Boyer (2012). Whereas these equations generate quantitative estimates of frequency sensitivity in Euarchonta, no members of Glires were included in the original sample. Therefore, while we employ these relationships, the quantitative results should be treated as indicative. We assumed the SPL at 250 Hz as a threshold for measuring low-frequency sensitivity and SPL at 32 kHz for high-frequency sensitivity after Coleman and Boyer (2012). High-and low-frequency thresholds are measured in decibels (dB) and indicate how sensitive an animal's hearing is relative to another. A lower threshold is indicative of more sensitivity to a particular hearing frequency compared with a higher threshold.

| Structure of the bony labyrinth
The morphology of the cochlea and SCCs in Megalagus resembles closely that in Palaeolagus (see Ruf et al., 2021), differing slightly in the SCCs shape and their spatial arrangement. The cochlea of Megalagus is tightly coiled and conic, but relatively flat; it has two turns approximately and is a bit shorter than that of Palaeolagus (Ruf et al., 2021). The ASC in Megalagus has the largest radius (ASR = 1.96 mm; Table 1; see Appendix A) of the three canals, similar to Palaeolagus (Ruf et al., 2021), modern lagomorphs, other Glires (including Rhombomylus; see Meng et al., 2003), and plesiadapiforms (Silcox et al., 2009). Interestingly, the PSC of Megalagus has the shortest radius, in contrast to other lagomorphs (e.g., Lepus arcticus or Ochotona pallasi) as well as to Rhombomylus, in which the shortest radius is found for the LSC. Compared with Palaeolagus, the canals in Megalagus have a more regular (almost ideally circular) course, while in the former they are slightly compressed either laterally, anteriorly, or posteriorly.
Similar to Palaeolagus haydeni, Megalagus turgidus differs significantly from crown lagomorphs in exhibiting a secondary common crus, a structure absent in extant lagomorphs and regarded as plesiomorphic (see Ruf et al., 2021). Its presence derives from the relative position of the LSC with respect to the PSC, where the inferior end of the latter reaches as far down as the plane defined by the LSC and meets the posterior end of the LSC, causing them to have a common course for a short distance and share also the hollow space containing the posterior ampulla ( Figure 1). In modern lagomorphs, the inferior end of the PSC extends much lower than the plane defined by the LSC, which goes into the vestibule separately, thereby not forming a unified secondary common crus (Ekdale, 2013;Ruf et al., 2021).

The round window (fenestra cochleae) in Megalagus turgidus
does not extend posteriorly beyond the PSC, similar to Palaeolagus (Ruf et al., 2021) Spoor et al. (2007) observed that more agile animals tend to have larger radii of the SCCs for a given body mass in a sample of 210 living mammal species including two leporids (Lepus europaeus and Oryctolagus cunicu lus). They identified "agility" with speed, and the analyzed species were grouped into six agility categories. Our sample includes only the Glires from Spoor et al. (2007), which are categorized as slow (2), medium (4), and fast (6). No Glires were represented for the extremely slow (1) and medium-slow (3) categories; the medium-fast (4) category was made up exclusively of the two leporid species included in Spoor et al. (2007).

| Locomotor agility
Our more extensive lagomorph sample (including rabbits, hares, and extant pikas [Ochotona], as well as the extinct Megalagus) better captures the diversity of the group. The results show that the ochotonids, small (150-250 g) and rather slow lagomorphs, and the smallest living leporid Brachylagus (Smith et al., 2018) have higher inferred agility scores than the larger leporids ( Figure 2). The latter group is known for their excellent cursorial abilities, especially well-expressed in true hares (Lepus). Such results suggest that linear speed and maneuverability, although closely related, are quite different phenomena. Agility can be considered in terms of the frequency and erraticism of head movement (Jeffery & Cox, 2010). These are functionally related not only to fast locomotion but also to quick response to visual cues. In lagomorphs, our conclusion is supported further by behavioral clues: pikas that inhabit mostly the rocky habitat of high mountains (talus patches) or semidesert mountain foothills are constantly challenged by their environment to move swiftly among boulders, climbing unstable substrates, and squeezing through crevices. Such locomotion requires high maneuverability and quick response to surface changes. Furthermore, the pygmy rabbit (Brachylagus idahoensis), the only leporid showing an unexpectedly high agility score (Figure 2) is at the same time the only leporid which does not leap effectively, but rather hops quickly, zigzagging in dense sagebrush cover (Green & Flinders, 1980).  "Agility score" based on LSR and agility category according to Spoor et al. (2007) are 3.59 and "medium," respectively.

| Hearing range
The hearing sensitivity of Megalagus reconstructed for the low- which are generally less sensitive than modern ochotonids in this respect (29.78-33.63 dB; Figure 3; see Appendix A for details). On the contrary, SPL reconstructed at 32 kHz indicates that Megalagus perceived high-frequency sounds at 12.08 dB, which makes this species more sensitive than pikas (13.83-28.58 dB) and all analyzed hares (12.77-27.16 dB) but was less sensitive than rabbits (10.72 dB; Figure 3).
Only a handful of studies exists on behaviorally tested hearing sensitivity in Lagomorpha, which allows us to compare our estimates with actual values. According to Heffner and Masterton (1980), the low-frequency sensitivity for Oryctolagus is 39.5 dB, which agrees with our estimate of 40.7 dB, and the high-frequency sensitivity is 20-26 dB versus predicted 10.7 dB (Figure 3; Table A4). The Eastern cottontail (Sylvilagus floridanus) exhibits low-frequency sensitivity of 67-77 dB (Heffner et al., 2020) versus predicted 57.9 dB (Table A4), and high-frequency sensitivity ~20 dB (Heffner et al., 2020) versus predicted 6.3 dB (Table A4). Thus, the discrepancies between experimental and estimated data are about 10 dB. This is comparable to the intraspecific range of variation for rodents, for example, Cynomys ludovicianus (measured low-frequency sensitivity 25-36.5 dB, highfrequency sensitivity 75-over 92 dB; Heffner et al., 1994), or guinea pig (measured low-frequency sensitivity 25-46 dB, high-frequency sensitivity 5-16 dB; Heffner et al., 1971).

| DISCUSS ION
The agility score and dimensions of the inner ear structures (see Appendix A for details) of Megalagus turgidus are more similar to those of extant leporids such as Lepus americanus or L. arcticus.
These two last species are not noted for their great agility. They inhabit boreal regions of North America, covered by taiga or tundra type of vegetation, and having a deep snow cover during the winter.
These factors hamper both fast locomotion and maneuverability, although the Arctic hare is known for its endurance to cover long distances (Lai et al., 2022). In the case of Megalagus, the reconstructed environment of the early Oligocene Brule Formation indicates open woodland habitat with abundant bushes and restricted grassy and herbaceous openings (Hutchinson, 1989;Leopold et al., 1992;Retallack, 1983), which would have been similar to the habitat of extant Lepus americanus. Dawson (1958) considered the postcranial evidence in Megalagus turgidus and concluded that this species was not a rapid cursor and may have had a similar locomotor behavior to that of present-day pikas (Ochotona).
With respect to the SCC proportions, Schmelzle et al. (2007) observed that in marsupial species which stand in a more erect posture,

F I G U R E 2 Relationship between body mass (BM) and lateral semicircular canal radius (LSR) for extant lagomorphs and rodents, and
Megalagus. Megalagus marked with yellow asterisk. Linear ordinary least squares regression is based on raw data in this paper (lagomorphs; see Appendix A), Ekdale (2013; Lepus californicus and Sylvilagus floridanus), and Spoor et al. (2007;rodents). For simplification, our "medium" category designation includes categories 3 ("medium slow") and 4 ("medium") of Spoor et al. (2007).
like kangaroos and wallabies (genus Macropus), the ASC is much taller than the PSC in comparison with species with a rather horizontal posture, in which the ASC and the PSC had a similar height.
Interestingly, all lagomorphs also have the ASC generally taller than the PSC, especially leporids. However, Megalagus has a similar ASCto-PSC ratio to ochotonids, which display a more uniform height between the anterior and posterior SCCs. Leporids do not routinely exhibit an erect posture, contrasting in this way from Macropus, for example, but they do share to certain extent a saltatorial (although not ricochetal) type of locomotion with kangaroos and wallabies.
On the contrary, ochotonids and Megalagus do not share the typical leaping-gallop locomotion of leporids, having a rather ambulatory locomotion. Also, a higher ASC with respect to the PSC is observed in Leptictidium, a saltatorial leptictidan, compared with Leptictis and Palaeoictops, nonsaltatorial leptictidans (Ruf et al., 2016). Therefore, a proportionally taller ASC with respect to the PSC may be associated with saltatorial locomotion, that is, fast and repetitive movements of the head (and body) along the vertical axis.
The estimated hearing sensitivity supports lagomorphs as better adapted to high-frequency sounds, because all lagomorph species including Megalagus turgidus show lower SPLs for the high frequencies than for the low ones ( Figure 3). However, our results do not fully confirm previous observations that smaller mammals have heightened high-frequency sensitivity, which was inferred to have been mainly to take advantage of spectral cues that aid in the ability to localize the source of sound (Heffner et al., 2020;Heffner & Heffner, 2010).
Ochotonids are smaller than leporids, but they are not among the species with greater hearing sensitivity to high frequencies. Ochotona princeps is the least sensitive to high-frequency sounds of our entire lagomorph sample (Figure 3). This reversed pattern might be explained by the fact that ochotonids have a more complex vocalization repertoire than leporids, perform calls with a wide frequency F I G U R E 3 Comparisons of hearing sensitivity of Megalagus to extant lagomorphs. The estimations based on predicted low-frequency and high-frequency hearing sensitivity. The SPL at 250 Hz (SPL 250Hz ; red) was used as a proxy for low-frequency sensitivity and sound pressure at 32 kHz (SPL 32kHz ; green) as a proxy for high-frequency sensitivity. The lower the sound pressure is, the more increased is the sensitivity. Actual data from behavioral audiograms for Oryctolagus cuniculus (from Heffner & Masterton, 1980) and Sylvilagus floridanus (from Heffner et al., 2020) in light red (SPL 250Hz ) and light green (SPL 32kHz ), respectively. Some ecological and behavioral traits (social and burrowing behavior, and preferred landscape) marked on the chart for the particular groups: pikas (Ochotona; yellow), Megalagus (orange), rabbits (light green), and hares (Lepus; dark green). Qualitative data on extant lagomorphs from Smith et al. (2018).
range (Konishi, 1970;Trefry & Hik, 2010) and, in some populations, even produce multiple-note calls (Conner, 1982). Moreover, highfrequency calls in Altai pikas (Ochotona alpina) are within the range of 7.31-15.46 kHz (Volodin et al., 2018), which is probably similar to that of other pikas, and much less than 32 kHz used for high-frequency sensitivity estimation. Although there is an overlap between leporids and ochotonids in their high-frequency hearing sensitivity, ochotonids display higher low-frequency sensitivity than leporids, which can be related to a more open-landscape habitat of the former, where the low-frequency sounds propagate easier and for longer distances.
Our results show that early lagomorphs including Megalagus were more leporid-like in terms of hearing sensitivity, and accord-

CO N FLI C T O F I NTE R E S T S TATE M E NT
We declare no conflict of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
All morphometric data that originated as a result of this study are available in Appendix A.